Molecular Methods

Protein-Based Technique

 

lsozyme analyses

Fatty acid profiles


MALDI-TOF mass spectrometry

Molecular methods to characterize, identify, and classify organisms do not depend on the subjective judgment of a human being as it might occur us-ing classical methods, but are based on the objective information (molecules) deriving from the target organism. Thus, molecular methods are increasingly used to identify organisms and for taxonomy research (molecular systematic). In the 1980s, molecular methods were established for wood-decay and staining fungi. Mainly, the fungal proteins (enzymes) and nucleic acids are used. It is outside the intention of this book to describe all molecular techniques that are currently used in the field of biology. The following overview comprises only some methods and results that are related to the characterization, identifica-tion, and phylogeny of wood-inhabiting fungi, particularly wood-decay fungi. Genome sequencing (meanwhile over 100 genomes are sequenced), molecular engineering, cloning, etc. are briefly addressed in other chapters. As an example of the latter, Lee et al. (2002) transformed the wild-type and the albino strain of the blue-stain fungus Ophiostoma piliferum with a green fluorescent protein (GFP) to microscopically differentiate the GPF-expressing fungi from other fungi in wood.

 

SDS polyacrylamide gel electrophoresis( SDS-PAGE) In SDS-PAGE, the whole cell protein is extracted from fungal tissue, denatured, and negatively charged with mercaptoethanol and sodium dodecyl sulfate !d by (SDS). The proteins are separated according to size on acrylamide gels and ernet visualized by Coomassie blue, amido black, fast green, imidazole-zinc or silver Eu- staining. The banding pattern obtained discriminates at the species level and nisa- slightly below. tions SDS-PAGE was used for wood-inhabiting Ascomycetes and Deuteromycetes .e re- like the Cancer stain disease fungus of plane, Ceratocystis fimbriata f. platani, (Granata et al. 1992) and Trichoderma species (Wallace et al. 1992). The technique also differentiated a number of wood-decay fungi (Schmidt and Kebernik 1989; Vigrow et al. 1989, 1991a; Schmidt and Moreth-Kebernik Df mi-1991a, 1993; Palfreyman et al. 1991; McDowell et al. 1992; Schmidt and Moreth 1995). For example, the closely related Serpula lacrymans, S. himantioides and the “American dry rot fungus”, Meruliporia incrassata, were distinguished (Schmidt and Moreth-Kebernik 1989a). That the technique also detected a misnamed isolate of S. lacrymans. In addition, monokaryons and F1 dikaryons of S. lacrymans exhibited the typical species profile (Schmidt and Moreth-Kebernik 1990). There was no need to extrapolate on a possible influence of culture age or medium composition (Schmidt and Kebernik 1989).

 

SDS-PAGE is fast when the sample originates from a pure culture and can be performed within 1 day. Reproducible homemade gels require accuracy and precautions, as acrylamide is carcinogenic in the unpolymerized form. Prefabricated gels are expensive. At least regarding wood-decay fungi, the method did not reach a practical application.

 

Isozyme analyses have been used to distinguish similar and closely related species and forms, for investigations on the genetical variability and on the spread of pathogens (e.g., Blaich and Esser 1975; Prillinger and Molitoris 1981; Micales et al. 1992). Being functional proteins, isozymes are investigated by native electrophoresis or isoelectric focusing. There are a number of investi-gations on mycorrhizal fungi, e.g., Pisolithus and Scleroderma species (Sims et al. 1999) and on tree parasites, like Armillaria species (Bragaloni et al. 1997) and Heterobasidion annosum (Karlsson and Stenlid 1991). Two-dimensional gel electrophoresis, comprising isoelectric focusing and subsequent SDS-PAGE, is able to separate a sample of a large number of proteins.

 

Immunological methods Wood fungi can be also detected and identified by immunological (serolog-ical) methods. Immunological assays use polyclonal antisera or monoclonal antibodies. Antisera produced by animals like mice and rabbits as answer to the injection of mycelial fragments, extracts or culture filtrates are inves-tigated by Western blotting, enzyme-linked immunosorbent assay (ELISA) or immunofluorescence (Clausen 2003).

However, the experiments often ex-hibit cross-reactions with non-target organisms, even when monoclonal anti-bodies after fusion with myeloma cells (hybridomas) are used. Investigations were performed with e.g., Armillaria spp., Coniophora puteana, Gloeophyllum trabeum, Lentinula edodes, Lentinus lepideus, Oligoporus placenta, Phellinus pini, S. lacrymans, Trametes versicolor and with wood-stain fungi (Jellison and Goodell 1988; Palfreyman et al. 1988; Breuil et al. 1988; Glancy et al. 1990; Burdsall et al. 1990; Vigrow et al. 1991b, 1991c; Clausen et al. 1991, 1993; Kim et al. 1991a, 1991b, 1993; Toft 1992, 1993; McDowell et al. 1992; Clausen 1997a; Breuil and Seifert 1999; Hunt et al. 1999). The diagnostic potential lies in the identification of species without the need of preceding isolation and pure culturing and in the detection of fungi at early stages of decay (Clausen and Kartal 2003).
The methods may become applicable when the producing techniques for hybridomas and diagnostic kits have been established. Immunological methods were also used to visualize the distribution of enzymes of wood-degrading fungi within and around the hypha and in woody tissue.

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